Browsing by Subject "Receptors, Cell Surface"
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Item Open Access An entirely cell-based system to generate single-chain antibodies against cell surface receptors.(2008) Chen, Yu-Hsun JasonThe generation of recombinant antibodies (Abs) using phage display is a proven method to obtain a large variety of Abs that bind with high affinity to a given antigen (Ag). Traditionally, the generation of single chain Abs depends on the use of recombinant proteins in several stages of the procedure. This can be a problem, especially in the case of cell surface receptors, because Abs generated and selected against recombinant proteins may not bind the same protein expressed on a cell surface in its native form and because the expression of some receptors as recombinant proteins is problematic. To overcome these difficulties, we developed a strategy to generate single chain Abs that does not require the use of purified protein at any stage of the procedure. In this strategy, stably transfected cells are used for the immunization of mice, measuring Ab responses to immunization, panning the phage library, high throughputs creening of arrayed phage clones, and characterization of recombinant single chain variable regions(scFvs). This strategy was used to generate a panel of single chain Abs specific for the innate immunity receptor Toll‐like receptor2 (TLR2). Once generated, individual scFvs were subcloned into an expression vector allowing the production of recombinant antibodies in insect cells, thus avoiding the contamination of recombinant Abs with microbial products. This cell‐based system efficiently generates Abs that bind native molecules displayed on cell surfaces, bypasses the requirement of recombinant protein production, and avoids risks of microbial component contamination. However, an inconvenience of this strategy is that it requires construction of a new library for each target TLR. This problem might be solved by using non‐immune antibody libraries to obtain antibodies against multiple TLRs. Non‐immune libraries contain a wide variety of antibodies but these are often low affinity, while immune libraries, derived from immunized animals, containa high frequency of high affinity antibodies, but are typically limited to a single antigen. In addition, it can be difficult to produce non‐immune libraries with sufficient complexity to select Abs against multiple Ags. Because the re‐assortment of VH and VL regions that occurs during antibody library construction greatly increases library complexity, we hypothesized that an immune antibody library produced against one member of a protein family would contain antibodies specific for other members of the same protein family. Here, we tested this hypothesis by mining an existing anti‐hTLR2 antibody library for antibodies specific for other members of the TLR family. This procedure, which we refer to as homolog mining, proved to be effective. Using a cell‐based system to pan and screen our anti‐hTLR2 library, we identified single chain antibodies specific for three of the four hTLR2 homologs we targeted. The antibodies identified, anti‐murine TLR2, anti‐hTLR5, and anti‐hTLR6, bind specifically to their target, with no cross‐reactivity to hTLR2 or other TLRs tested. These results demonstrate that combinatorial re‐assortment of VH and VL fragments during Ab library construction increases Ab repertoire complexity, allowing antibody libraries produced by immunization with one antigen to be used to obtain antibodies specific to related antigens. The principle of homolog mining may be extended to other protein families and will facilitate and accelerate antibody production processes. In conclusion, we developed an entirely cell‐based method to generate antibodies that bind to native molecules on the cell surface, while eliminating the requirement of recombinant proteins and the risk of microbial component contamination. With homolog mining, this system is capable of generating antibodies not only against the original immunized Ag, but also against homologous Ags. In combination, this system proved to be an effective and efficient means for generating multiple antibodies that bind to multiple related Ags as they are displayed on cell surfaces.Item Open Access Analysis of the mouse transcriptome for genes involved in the function of the nervous system.(Genome Res, 2003-06) Gustincich, Stefano; Batalov, Serge; Beisel, Kirk W; Bono, Hidemasa; Carninci, Piero; Fletcher, Colin F; Grimmond, Sean; Hirokawa, Nobutaka; Jarvis, Erich D; Jegla, Tim; Kawasawa, Yuka; LeMieux, Julianna; Miki, Harukata; Raviola, Elio; Teasdale, Rohan D; Tominaga, Naoko; Yagi, Ken; Zimmer, Andreas; Hayashizaki, Yoshihide; Okazaki, Yasushi; RIKEN GER Group; GSL MembersWe analyzed the mouse Representative Transcript and Protein Set for molecules involved in brain function. We found full-length cDNAs of many known brain genes and discovered new members of known brain gene families, including Family 3 G-protein coupled receptors, voltage-gated channels, and connexins. We also identified previously unknown candidates for secreted neuroactive molecules. The existence of a large number of unique brain ESTs suggests an additional molecular complexity that remains to be explored.A list of genes containing CAG stretches in the coding region represents a first step in the potential identification of candidates for hereditary neurological disorders.Item Open Access Brief Glutamine Pretreatment Increases Alveolar Macrophage CD163/Heme Oxygenase-1/p38-MAPK Dephosphorylation Pathway and Decreases Capillary Damage but Not Neutrophil Recruitment in IL-1/LPS-Insufflated Rats.(PLoS One, 2015) Fernandez-Bustamante, Ana; Agazio, Amanda; Wilson, Paul; Elkins, Nancy; Domaleski, Luke; He, Qianbin; Baer, Kaily A; Moss, Angela FD; Wischmeyer, Paul E; Repine, John EBACKGROUND: Glutamine (GLN) attenuates acute lung injury (ALI) but its effect on alveolar macrophages is unknown. We hypothesized that GLN pretreatment would induce the anti-inflammatory CD163/heme oxygenase (HO)-1/p38-MAPK dephosphorylation pathway in alveolar macrophages and reduce ALI in rats insufflated with interleukin-1 (IL-1) and lipopolysaccharide (LPS). METHODS: Male Sprague-Dawley rats were randomized to the following groups: GLN-IL-1/LPS-, GLN+IL-1/LPS-, GLN-IL-1/LPS+, and GLN+IL-1/LPS+. GLN pretreatment was given via gavage (1 g/kg L-alanyl-L-glutamine) daily for 2 days. ALI was subsequently induced by insufflating 50 ng IL-1 followed by 5mg/kg E.coli LPS. After 24h, bronchoalveolar lavage (BAL) protein, lactate dehydrogenase (LDH) and neutrophil concentrations were analyzed. BAL alveolar macrophage CD163+ expression, HO-1 and p38-MAPK concentrations were measured, as well as alveolar macrophage tumor necrosis factor (TNF)-α and interleukin (IL)-10 concentrations. Histology and immunofluorescence studies were also performed. RESULTS: Following IL-1/LPS insufflation, GLN pretreated rats had significantly decreased BAL protein and LDH concentrations, but not BAL neutrophil counts, compared to non-GLN pretreated rats. The number of alveolar macrophages and the number of CD163+ macrophages were significantly increased in GLN pretreated IL-1/LPS-insufflated rats compared to non-GLN pretreated, IL-1/LPS-insufflated rats. GLN pretreatment before IL-1/LPS also significantly increased HO-1 concentrations and dephosphorylated p38-MAPK levels but not cytokine levels in alveolar macrophages. Immunofluorescence localized CD163 and HO-1 in alveolar macrophages. CONCLUSION: Short-term GLN pretreatment activates the anti-inflammatory CD163/HO-1/p38-MAPK dephosphorylation pathway of alveolar macrophages and decreases capillary damage but not neutrophil recruitment in IL-1/LPS-insufflated rats.Item Open Access Characterization of the murine BEK fibroblast growth factor (FGF) receptor: activation by three members of the FGF family and requirement for heparin.(Proc Natl Acad Sci U S A, 1992-04-15) Mansukhani, A; Dell'Era, P; Moscatelli, D; Kornbluth, S; Hanafusa, H; Basilico, CThe bek gene encodes a member of the high-affinity fibroblast growth factor receptor family. The BEK/FGFR-2 receptor is a membrane-spanning tyrosine kinase with the typical features of FGF receptors. We have cloned a murine bek cDNA and expressed it in receptor-negative Chinese hamster ovary cells and in 32D myeloid cells. The BEK receptor expressed in Chinese hamster ovary cells binds acidic FGF, basic FGF, and Kaposi FGF equally well but does not bind keratinocyte growth factor or FGF-5 appreciably. Upon treatment with basic FGF or Kaposi FGF, the BEK receptor is phosphorylated and a mitogenic response is achieved. Heparan sulfate proteoglycans have been shown to play an obligate role in basic FGF binding to the high-affinity FLG receptor. Unlike the BEK-expressing Chinese hamster ovary cells, 32D cells expressing the BEK receptor require the addition of exogenous heparin in order to grow in the presence of basic FGF or Kaposi FGF. We show that the addition of heparin greatly enhances the binding of radio-labeled basic FGF to the receptor. Thus the BEK receptor, like FLG, also requires an interaction with heparan sulfate proteoglycans to facilitate binding to its ligands.Item Open Access Direct evidence that Gi-coupled receptor stimulation of mitogen-activated protein kinase is mediated by G beta gamma activation of p21ras.(Proc Natl Acad Sci U S A, 1994-12-20) Koch, WJ; Hawes, BE; Allen, LF; Lefkowitz, RJStimulation of Gi-coupled receptors leads to the activation of mitogen-activated protein kinases (MAP kinases). In several cell types, this appears to be dependent on the activation of p21ras (Ras). Which G-protein subunit(s) (G alpha or the G beta gamma complex) primarily is responsible for triggering this signaling pathway, however, is unclear. We have demonstrated previously that the carboxyl terminus of the beta-adrenergic receptor kinase, containing its G beta gamma-binding domain, is a cellular G beta gamma antagonist capable of specifically distinguishing G alpha- and G beta gamma-mediated processes. Using this G beta gamma inhibitor, we studied Ras and MAP kinase activation through endogenous Gi-coupled receptors in Rat-1 fibroblasts and through receptors expressed by transiently transfected COS-7 cells. We report here that both Ras and MAP kinase activation in response to lysophosphatidic acid is markedly attenuated in Rat-1 cells stably transfected with a plasmid encoding this G beta gamma antagonist. Likewise in COS-7 cells transfected with plasmids encoding Gi-coupled receptors (alpha 2-adrenergic and M2 muscarinic), the activation of Ras and MAP kinase was significantly reduced in the presence of the coexpressed G beta gamma antagonist. Ras-MAP kinase activation mediated through a Gq-coupled receptor (alpha 1-adrenergic) or the tyrosine kinase epidermal growth factor receptor was unaltered by this G beta gamma antagonist. These results identify G beta gamma as the primary mediator of Ras activation and subsequent signaling via MAP kinase in response to stimulation of Gi-coupled receptors.Item Open Access Heptahelical receptor signaling: beyond the G protein paradigm.(J Cell Biol, 1999-05-31) Hall, RA; Lefkowitz, Robert J; Premont, Richard ThomasItem Open Access Human-chimpanzee differences in a FZD8 enhancer alter cell-cycle dynamics in the developing neocortex.(Curr Biol, 2015-03-16) Boyd, J Lomax; Skove, Stephanie L; Rouanet, Jeremy P; Pilaz, Louis-Jan; Bepler, Tristan; Gordân, Raluca; Wray, Gregory A; Silver, Debra LThe human neocortex differs from that of other great apes in several notable regards, including altered cell cycle, prolonged corticogenesis, and increased size [1-5]. Although these evolutionary changes most likely contributed to the origin of distinctively human cognitive faculties, their genetic basis remains almost entirely unknown. Highly conserved non-coding regions showing rapid sequence changes along the human lineage are candidate loci for the development and evolution of uniquely human traits. Several studies have identified human-accelerated enhancers [6-14], but none have linked an expression difference to a specific organismal trait. Here we report the discovery of a human-accelerated regulatory enhancer (HARE5) of FZD8, a receptor of the Wnt pathway implicated in brain development and size [15, 16]. Using transgenic mice, we demonstrate dramatic differences in human and chimpanzee HARE5 activity, with human HARE5 driving early and robust expression at the onset of corticogenesis. Similar to HARE5 activity, FZD8 is expressed in neural progenitors of the developing neocortex [17-19]. Chromosome conformation capture assays reveal that HARE5 physically and specifically contacts the core Fzd8 promoter in the mouse embryonic neocortex. To assess the phenotypic consequences of HARE5 activity, we generated transgenic mice in which Fzd8 expression is under control of orthologous enhancers (Pt-HARE5::Fzd8 and Hs-HARE5::Fzd8). In comparison to Pt-HARE5::Fzd8, Hs-HARE5::Fzd8 mice showed marked acceleration of neural progenitor cell cycle and increased brain size. Changes in HARE5 function unique to humans thus alter the cell-cycle dynamics of a critical population of stem cells during corticogenesis and may underlie some distinctive anatomical features of the human brain.Item Open Access Life is sweet: the cell biology of glycoconjugates.(Molecular biology of the cell, 2019-03) Broussard, Alex C; Boyce, MichaelCells are dazzling in their diversity, both within and across organisms. And yet, throughout this variety runs at least one common thread: sugars. All cells on Earth, in all domains of life, are literally covered in glycans, a term referring to the carbohydrate portion of glycoproteins and glycolipids. In spite of (or, perhaps, because of) their tremendous structural and functional complexity, glycans have historically been underexplored compared with other areas of cell biology. Recently, however, advances in experimental systems and analytical methods have ushered in a renaissance in glycobiology, the study of the biosynthesis, structures, interactions, functions, and evolution of glycans. Today, glycobiology is poised to make major new contributions to cell biology and become more fully integrated into our understanding of cell and organismal physiology.Item Open Access Mechanisms of membrane-receptor regulation. Biochemical, physiological, and clinical insights derived from studies of the adrenergic receptors.(N Engl J Med, 1984-06-14) Lefkowitz, RJ; Caron, MG; Stiles, GLItem Open Access Mitogenic signaling via G protein-coupled receptors.(Endocr Rev, 1996-12) van Biesen, T; Luttrell, LM; Hawes, BE; Lefkowitz, RJItem Open Access Monoclonal antibodies reveal receptor specificity among G-protein-coupled receptor kinases.(Proc Natl Acad Sci U S A, 1996-07-23) Oppermann, M; Diversé-Pierluissi, M; Drazner, MH; Dyer, SL; Freedman, NJ; Peppel, KC; Lefkowitz, RJGuanine nucleotide-binding regulatory protein (G protein)-coupled receptor kinases (GRKs) constitute a family of serine/threonine kinases that play a major role in the agonist-induced phosphorylation and desensitization of G-protein-coupled receptors. Herein we describe the generation of monoclonal antibodies (mAbs) that specifically react with GRK2 and GRK3 or with GRK4, GRK5, and GRK6. They are used in several different receptor systems to identify the kinases that are responsible for receptor phosphorylation and desensitization. The ability of these reagents to inhibit GRK- mediated receptor phosphorylation is demonstrated in permeabilized 293 cells that overexpress individual GRKs and the type 1A angiotensin II receptor. We also use this approach to identify the endogenous GRKs that are responsible for the agonist-induced phosphorylation of epitope-tagged beta2- adrenergic receptors (beta2ARs) overexpressed in rabbit ventricular myocytes that are infected with a recombinant adenovirus. In these myocytes, anti-GRK2/3 mAbs inhibit isoproterenol-induced receptor phosphorylation by 77%, while GRK4-6-specific mAbs have no effect. Consistent with the operation of a betaAR kinase-mediated mechanism, GRK2 is identified by immunoblot analysis as well as in a functional assay as the predominant GRK expressed in these cells. Microinjection of GRK2/3-specific mAbs into chicken sensory neurons, which have been shown to express a GRK3-like protein, abolishes desensitization of the alpha2AR-mediated calcium current inhibition. The intracellular inhibition of endogenous GRKs by mAbs represents a novel approach to the study of receptor specificities among GRKs that should be widely applicable to many G-protein-coupled receptors.Item Open Access Multiple endocytic pathways of G protein-coupled receptors delineated by GIT1 sensitivity.(Proc Natl Acad Sci U S A, 2000-02-01) Claing, A; Perry, SJ; Achiriloaie, M; Walker, JK; Albanesi, JP; Lefkowitz, RJ; Premont, RTRecently, we identified a GTPase-activating protein for the ADP ribosylation factor family of small GTP-binding proteins that we call GIT1. This protein initially was identified as an interacting partner for the G protein-coupled receptor kinases, and its overexpression was found to affect signaling and internalization of the prototypical beta(2)-adrenergic receptor. Here, we report that GIT1 overexpression regulates internalization of numerous, but not all, G protein-coupled receptors. The specificity of the GIT1 effect is not related to the type of G protein to which a receptor couples, but rather to the endocytic route it uses. GIT1 only affects the function of G protein-coupled receptors that are internalized through the clathrin-coated pit pathway in a beta-arrestin- and dynamin-sensitive manner. Furthermore, the GIT1 effect is not limited to G protein-coupled receptors because overexpression of this protein also affects internalization of the epidermal growth factor receptor. However, constitutive agonist-independent internalization is not regulated by GIT1, because transferrin uptake is not affected by GIT1 overexpression. Thus, GIT1 is a protein involved in regulating the function of signaling receptors internalized through the clathrin pathway and can be used as a diagnostic tool for defining the endocytic pathway of a receptor.Item Open Access Neuronal IL-4Rα modulates neuronal apoptosis and cell viability during the acute phases of cerebral ischemia.(The FEBS journal, 2018-08) Lee, Han Kyu; Koh, Sehwon; Lo, Donald C; Marchuk, Douglas AIschemic stroke caused by an embolus or local thrombosis results in neural tissue damage (an infarct) in the territory of the occluded cerebral artery. Decades of studies have increased our understanding of the molecular events during cerebral infarction; however, translation of these discoveries to druggable targets for ischemic stroke treatment has been largely disappointing. Interleukin-4 (IL-4) is a multifunctional cytokine that exerts its cellular activities via the interleukin-4 receptor α (IL-4Rα). This cytokine receptor complex is associated with diverse immune and inflammatory responses. Recent studies have suggested a role of the cytokine IL-4 in long-term ischemic stroke recovery, involving immune cell activity. In contrast, the role of the receptor, IL-4Rα especially in the acute phase of infarction is unclear. In this study, we determined that IL-4Rα is expressed on neurons and that during the early phases of cerebral infarction (24 h) levels of this receptor are increased to regulate cellular apoptosis factors through activation of STAT6. In this context, we show a neuroprotective role for IL-4Rα in an in vivo surgical model of cerebral ischemia and in ex vivo brain slice explants, using both genetic knockout of this receptor and RNAi-mediated gene knockdown. IL-4Rα may therefore represent a novel target and pathway for therapeutic development in ischemic stroke.Item Open Access NgBR is essential for endothelial cell glycosylation and vascular development.(EMBO Rep, 2016-02) Park, Eon Joo; Grabińska, Kariona A; Guan, Ziqiang; Sessa, William CNgBR is a transmembrane protein identified as a Nogo-B-interacting protein and recently has been shown to be a subunit required for cis-prenyltransferase (cisPTase) activity. To investigate the integrated role of NgBR in vascular development, we have characterized endothelial-specific NgBR knockout embryos. Here, we show that endothelial-specific NgBR knockout results in embryonic lethality due to vascular development defects in yolk sac and embryo proper. Loss of NgBR in endothelial cells reduces proliferation and promotes apoptosis of the cells largely through defects in the glycosylation of key endothelial proteins including VEGFR2, VE-cadherin, and CD31, and defective glycosylation can be rescued by treatment with the end product of cisPTase activity, dolichol phosphate. Moreover, NgBR functions in endothelial cells during embryogenesis are Nogo-B independent. These data uniquely show the importance of NgBR and protein glycosylation during vascular development.Item Open Access Phosphorylation of cell surface receptors: a mechanism for regulating signal transduction pathways.(Endocr Rev, 1988-02) Sibley, DR; Benovic, JL; Caron, MG; Lefkowitz, RJItem Open Access Regulation of G protein-coupled receptor signaling by scaffold proteins.(Circ Res, 2002-10-18) Hall, Randy A; Lefkowitz, Robert JThe actions of many hormones and neurotransmitters are mediated through stimulation of G protein-coupled receptors. A primary mechanism by which these receptors exert effects inside the cell is by association with heterotrimeric G proteins, which can activate a wide variety of cellular enzymes and ion channels. G protein-coupled receptors can also interact with a number of cytoplasmic scaffold proteins, which can link the receptors to various signaling intermediates and intracellular effectors. The multicomponent nature of G protein-coupled receptor signaling pathways makes them ideally suited for regulation by scaffold proteins. This review focuses on several specific examples of G protein-coupled receptor-associated scaffolds and the roles they may play in organizing receptor-initiated signaling pathways in the cardiovascular system and other tissues.Item Open Access Role of endocytosis in the activation of the extracellular signal-regulated kinase cascade by sequestering and nonsequestering G protein-coupled receptors.(Proc Natl Acad Sci U S A, 2000-02-15) Pierce, KL; Maudsley, S; Daaka, Y; Luttrell, LM; Lefkowitz, RJActing through a number of distinct pathways, many G protein-coupled receptors (GPCRs) activate the extracellular signal-regulated kinase (ERK)/mitogen-activated protein kinase (MAPK) cascade. Recently, it has been shown that in some cases, clathrin-mediated endocytosis is required for GPCR activation of the ERK/MAPK cascade, whereas in others it is not. Accordingly, we compared ERK activation mediated by a GPCR that does not undergo agonist-stimulated endocytosis, the alpha(2A) adrenergic receptor (alpha(2A) AR), with ERK activation mediated by the beta(2) adrenergic receptor (beta(2) AR), which is endocytosed. Surprisingly, we found that in COS-7 cells, ERK activation by the alpha(2A) AR, like that mediated by both the beta(2) AR and the epidermal growth factor receptor (EGFR), is sensitive to mechanistically distinct inhibitors of clathrin-mediated endocytosis, including monodansylcadaverine, a mutant dynamin I, and a mutant beta-arrestin 1. Moreover, we determined that, as has been shown for many other GPCRs, both alpha(2A) and beta(2) AR-mediated ERK activation involves transactivation of the EGFR. Using confocal immunofluorescence microscopy, we found that stimulation of the beta(2) AR, the alpha(2A) AR, or the EGFR each results in internalization of a green fluorescent protein-tagged EGFR. Although beta(2) AR stimulation leads to redistribution of both the beta(2) AR and EGFR, activation of the alpha(2A) AR leads to redistribution of the EGFR but the alpha(2A) AR remains on the plasma membrane. These findings separate GPCR endocytosis from the requirement for clathrin-mediated endocytosis in EGFR transactivation-mediated ERK activation and suggest that it is the receptor tyrosine kinase or another downstream effector that must engage the endocytic machinery.Item Open Access Role of ST2 in non-ST-elevation acute coronary syndrome in the MERLIN-TIMI 36 trial.(Clinical chemistry, 2012-01) Kohli, Payal; Bonaca, Marc P; Kakkar, Rahul; Kudinova, Anastacia Y; Scirica, Benjamin M; Sabatine, Marc S; Murphy, Sabina A; Braunwald, Eugene; Lee, Richard T; Morrow, David AObjective
We investigated the prognostic performance of ST2 with respect to cardiovascular death (CVD) and heart failure (HF) in patients with non-ST-elevation acute coronary syndrome (NSTE-ACS) in a large multinational trial.Background
Myocytes that are subjected to mechanical stress secrete ST2, a soluble interleukin-1 receptor family member that is associated with HF after STE-ACS.Methods
We measured ST2 with a high-sensitivity assay in all available baseline samples (N=4426) in patients enrolled in the Metabolic Efficiency With Ranolazine for Less Ischemia in the Non-ST-Elevation Acute Coronary Syndrome Thrombolysis in Myocardial Infarction 36 (MERLIN-TIMI 36), a placebo-controlled trial of ranolazine in NSTE-ACS. All events, including cardiovascular death and new or worsening HF, were adjudicated by an independent events committee.Results
Patients with ST2 concentrations in the top quartile (>35 μg/L) were more likely to be older and male and have diabetes and renal dysfunction. ST2 was only weakly correlated with troponin and B-type natriuretic peptide. High ST2 was associated with increased risk for CVD/HF at 30 days (6.6% vs 1.6%, P<0.0001) and 1 year (12.2% vs 5.2%, P<0.0001). The risk associated with ST2 was significant after adjustment for clinical covariates and biomarkers (adjusted hazard ratio CVD/HF 1.90, 95% CI 1.15-3.13 at 30 days, P=0.012; 1.51, 95% CI 1.15-1.98 at 1 year, P=0.003), with a significant integrated discrimination improvement (P<0.0001). No significant interaction was found between ST2 and ranolazine (Pinteraction=0.15).Conclusions
ST2 correlates weakly with biomarkers of acute injury and hemodynamic stress but is strongly associated with the risk of HF after NSTE-ACS. This biomarker and related pathway merit further investigation as potential therapeutic targets for patients with ACS at risk for cardiac remodeling.Item Open Access Salicylic acid receptors activate jasmonic acid signalling through a non-canonical pathway to promote effector-triggered immunity.(Nature communications, 2016-10-11) Liu, Lijing; Sonbol, Fathi-Mohamed; Huot, Bethany; Gu, Yangnan; Withers, John; Mwimba, Musoki; Yao, Jian; He, Sheng Yang; Dong, XinnianIt is an apparent conundrum how plants evolved effector-triggered immunity (ETI), involving programmed cell death (PCD), as a major defence mechanism against biotrophic pathogens, because ETI-associated PCD could leave them vulnerable to necrotrophic pathogens that thrive on dead host cells. Interestingly, during ETI, the normally antagonistic defence hormones, salicylic acid (SA) and jasmonic acid (JA) associated with defence against biotrophs and necrotrophs respectively, both accumulate to high levels. In this study, we made the surprising finding that JA is a positive regulator of RPS2-mediated ETI. Early induction of JA-responsive genes and de novo JA synthesis following SA accumulation is activated through the SA receptors NPR3 and NPR4, instead of the JA receptor COI1. We provide evidence that NPR3 and NPR4 may mediate this effect by promoting degradation of the JA transcriptional repressor JAZs. This unique interplay between SA and JA offers a possible explanation of how plants can mount defence against a biotrophic pathogen without becoming vulnerable to necrotrophic pathogens.Item Open Access The G-protein-coupled receptor phosphatase: a protein phosphatase type 2A with a distinct subcellular distribution and substrate specificity.(Proc Natl Acad Sci U S A, 1995-08-29) Pitcher, JA; Payne, ES; Csortos, C; DePaoli-Roach, AA; Lefkowitz, RJPhosphorylation of G-protein-coupled receptors plays an important role in regulating their function. In this study the G-protein-coupled receptor phosphatase (GRP) capable of dephosphorylating G-protein-coupled receptor kinase-phosphorylated receptors is described. The GRP activity of bovine brain is a latent oligomeric form of protein phosphatase type 2A (PP-2A) exclusively associated with the particulate fraction. GRP activity is observed only when assayed in the presence of protamine or when phosphatase-containing fractions are subjected to freeze/thaw treatment under reducing conditions. Consistent with its identification as a member of the PP-2A family, the GRP is potently inhibited by okadaic acid but not by I-2, the specific inhibitor of protein phosphatase type 1. Solubilization of the membrane-associated GRP followed by gel filtration in the absence of detergent yields a 150-kDa peak of latent receptor phosphatase activity. Western blot analysis of this phosphatase reveals a likely subunit composition of AB alpha C. PP-2A of this subunit composition has previously been characterized as a soluble enzyme, yet negligible soluble GRP activity was observed. The subcellular distribution and substrate specificity of the GRP suggests significant differences between it and previously characterized forms of PP-2A.